Rhabdomyosarcomas (RMS), according to the WHO classification, comprise three types of tumours sharing skeletal muscle differentiation while showing distinct genetic and biological features: 1) embryonal with a characteristic loss of heterozygocity at 11p15; 2) the more aggressive alveolar, most of which show pathognomonic Pax3 or Pax7-FKHR fusion genes due to the translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), respectively; and 3) the rare pleomorphic RMS with complex genetic aberrations and preferential occurrence in adults. As a group, RMS are the most frequent soft tissue tumour in children and have a poor prognosis. In particular, metastatic alveolar RMS in children more than 10 years old are often refractory to all current therapies including adjuvant bone marrow transplantation, resulting in 5-year survival rates of 5-20%. Immunotherapies or other (e.g. genetic) specific targeting strategies have not yet been applied due to the absence of a well defined RMS-specific gene target and the lack of suitable immunotherapeutic tools. However, it was shown previously that strong expression of fetal AChR mRNA and protein is virtually pathognomonic for non-innervated rhabdomyoblasts and rhabdomyotubes and, thus, for the vast majority of human embryonal and alveolar RMS; by contrast, fetal AChR expression is virtually absent in other tissues and in tumours without a rhabdomyoblast component.
The nicotinic AChR of skeletal muscle is a pentameric ion channel composed of five subunits. The fetal isoform is composed of 2α, 1β, 1γ ad 1δ-subunits, while the adult isoform exhibits an ε-subunit instead of the γ-subunit. During the later stages of development in uterus, the fetal AChR is gradually replaced by the adult isoform in virtually all innervated muscles. After birth, strong expression of fetal AChR is restricted to a few muscle fibres of unknown function occurring in extraoccular muscles and myoid cells of the thymus that are physiologically non-innervated muscle cells. However, high-level re-expression of fetal AChR can occur after birth following denervation, including denervating neuromuscular diseases.
In adult human innervated skeletal muscles, therefore, fetal AChR expression is exceedingly low. Only traces of γ-subunit mRNA can be detected by RT-PCR and RNAse protection assay, and very little by Northern blot. In addition, it has been shown that the mothers of newborns with arthrogryposis multiplex caused by the placental transfer of maternal antibodies specific to fetal AchR can be completely asymptomatic, suggesting that there is minimal expression of AChR γ-subunit protein on normal adult muscle.
Immunotoxins (ITs) are usually composed of a plant or bacterial toxin (like Pseudomonas exotoxin A=ETA) coupled to an antibody fragment for specific targeting of cells in the context of cancer, chronic inflammatory diseases, including autoimmune diseases and chronic infections, or transplantation. The antibody component of ITs is most often a murine Fab or Fv antibody fragment that has been humanised to minimize sensitization against the xenogeneic protein. Immunotoxins with a fully human antibody moiety (directed to HIV infected T-cells) have also been reported.
Impressive therapeutic efficacy of ITs has been demonstrated in a number of lymphomas and leukemias following short term application of the IT. By contrast, protracted use of an IT might be more adequate in poorly vascularized sarcomas. In such a clinical setting, is probably essential to minimize the sensitization against the mouse, rat or humanized antibody component of the IT.
Autoimmune disorders collectively cause much suffering and disability and many are particularly common in women of child-bearing age. The muscle weakness in the majority of myasthenia gravis (MG) patients results from autoantibodies to the acetylcholine receptor (AChR) which cause AChR loss, mainly by complement-mediated damage to the post-synaptic membrane and/or accelerated AChR degradation. The AChR consists of two α subunits and one β, γ and δ in the fetus. Together, these subunits form a cation-specific ion channel that opens when ACh binds simultaneously to the two sites at the α/δ and either the α/γ or α/ε interfaces. From about 30 weeks' gestation in humans, the ‘adult’-specific ε subunit gradually replaces the γ subunit, but the fetal isoform continues to be expressed on rare myoid cells in the adult thymic medulla.
Early-onset MG (before age 40; EOMG) shows a ˜3:1 female bias and a strong HLA-DR3-B8 association. The thymus is apparently an important site of autoimmunization; thymectomy is often beneficial and, in most EOMG cases, the thymus is invaded by lymph node-like T cell areas and germinal centers (GC, many of which show AChR-specificity. GC are well known sites of B cell memory generation and of antibody diversification by antigen-selection of somatic variants. Moreover, there is selective activation of thymic plasma cells spontaneously producing anti-AChR antibodies, with similar specificities to those in the patients' blood. The anti-AChR antibodies in typical MG patients are very heterogeneous and antibodies cloned from the thymus of typical EOMG patients show a range of specificities resembling those in the donors' sera.
About 8% of MG mothers have babies with transient neonatal MG, and their sera tend to have higher antibody titers against fetal than adult AChR, consistent with a role for thymic AChR in inducing their disease. In some rare examples, babies of MG mothers are born with severe developmental abnormalities, usually described as arthrogryposis multiplex congenita (AMC), which includes multiple joint contractures, hypoplasia of the lungs, other malformations and often fetal or neonatal death. The serum of these MG mothers, and others who are asymptomatic, contains antibodies that strongly and very selectively inhibit the ACh-triggered ion channel function of fetal AChR. These inhibitory antibodies persist in the maternal circulation and can transfer a similar condition to the pups after injection into pregnant mice. Interestingly, the first child of mothers of AMC babies is often unaffected and, when present, MG may not be clinically evident in the mother at the time of the first affected birth, suggesting that the maternal immune system may first be sensitized to AChR from the fetus. Moreover, the dominance of fetal AChR-specific antibodies in these women suggested that the B cell response might be clonally restricted.
Here we examined the relationship between inhibitory antibodies and parity in MG patients, and then took advantage of the fact that EOMG patients are treated by thymectomy, providing us with thymic B cells and plasma cells from which to clone and recombine fetal-AChR specific antibodies. We selected combinatorial Fabs from two MG mothers with high levels of fetal AChR-specific antibodies, whose babies had severe AMC. These Fabs proved to be strongly biased towards fetal AChR and were each dominated by one clone with extensive somatic diversification from an already highly mutated consensus sequence. These results suggest that the fetus could be responsible for immunizing the mother during pregnancy, with further diversification occurring subsequently in the thymus.